Tesi realitzada a l'Institut de Biologia Evolutiva (CSIC-UPF) / A major challenge in evolutionary biology is understanding the main drivers that underlie morphological and species diversity. Ecological opportunity—access to new or previously inaccessible niches—has been identified as one of the most important drivers of both phenotypic and species diversification. This is because the exploitation of new ecological niches is often accompanied by phenotypic differentiation among closely related taxa. This can in turn facilitate species diversification if phenotypic differentiation is associated with the appearance of reproductive isolation. The main goal of this thesis is to explore the extent in which a major source of ecological opportunity—the colonization of islands—have driven evolutionary diversification in different taxonomic and geographic contexts, specifically in the geckos of Arabia and the Socotra Archipelago and the Australasian diplodactyloid geckos. Island colonization is thought to provide a context of many available resources with few competitors and predators. This allows to colonizing groups the possibility to experience an “ecological release” and use a wider array of niches compared to their continental close-relatives. In such a situation, we expect an expansion of the morphospace in island groups typically associated with high rates of phenotypic and species diversification. We have found compelling evidence for this in both of the two mainland-island systems studied. The Australasian diplodactyloid geckos clearly expanded the range of phenotypic variation existing in the continent, producing the biggest and the smallest species in the radiation and were associated to accelerated rates of body size diversification compared to their closest relatives in the continent. Likewise, in the Hemidactylus geckos from Arabia-Socotra, island species were also associated to accelerated rates of phenotypic diversification and, as in the Australasian system, produced the most extreme sizes in the radiation. In this last mainland-island system, aside of reaching the maximum and the minimum sizes, the body size disparities in continental species assemblages were always significantly greater than the disparities computed by continental species assemblages. Nonetheless, when we compare Hemidactylus to other groups diversifying in the same islands, it appears that not all groups equally tend to diversify in body size. In fact, the comparison of the stages of diversification between the three gecko genera occurring in the Archipelago of Socotra revealed that not a single path of intra-island diversification was shared by all genera. Hemidactylus and Haemodracon greatly diversified in body size. However, in Pristurus diversification was strongly mediated by climatic shifts with size diversification being displaced to a subtle role. This is an important result as suggests that not all groups respond in the same way to similar amounts of ecological opportunity and that group-dependent (intrinsic) components can potentially play a role at defining the different stages of diversification. Aside of these examples of island diversification, in this thesis I also exposed remarkable examples of “island-like” patterns of diversification occurring in the continent. This was the case for the Australasian Pygopodidae, which attained rates of phenotypic diversification (possibly species diversification too) comparable to those found in island groups and was mostly mediated by the acquisition of a remarkable key innovation: a “snake-like” phenotype. Another remarkable example of continental diversification revealed in this thesis is a complex of highly divergent species existing within the subspecies Pristurus rupestris rupestris. This diversification took place in truly “island-like” setting, the Hajar Mountains in southeastern Arabia, formed by three main isolated blocks or “sky islands”. However, in this case diversification failed to take place within each of the “islands” and mostly was driven through intermittent pulses of dispersal and isolation taking place between two of the two main mountain blocks. Examples like this provide an example of how continental groups in some contexts, as the ones offered by mountain ranges, can fuel substantial amounts of diversification. In this case one of the greatest vertebrate diversification in Arabia. / Uno de los retos más importantes en biología evolutiva es entender las causas principales de la diversidad de fenotipos y de especies. La oportunidad ecológica (o el acceso a recursos nuevos y previamente inaccesibles) se considera como uno de los factores más importantes en la generación de nuevas especies y fenotipos. El objetivo principal de esta tesis es estudiar el papel generador de especies y fenotipos de uno de los grandes tipos de oportunidad ecológica, la colonización de islas. Para ello, se han usado dos sistemas continente-isla como modelo: los gecos de Arabia y el Archipiélago de Socotra y los gecos diplodactiloideos australasiáticos. Dado que las islas proporcionan ambientes con pocos competidores y depredadores, esto permite a los grupos insulares experimentar una gran expansión de nicho que puede estar asociada a una expansión del morfoespacio mediada por tasas elevadas de diversificación fenotípica y de especies. Los resultados de esta tesis respaldan totalmente esta hipótesis. Tanto en los gecos australasiáticos como en los gecos de Arabia y Socotra, la colonización de islas vino acompañada con aumentos sustanciales de disparidad fenotípica asociados con incrementos en las tasas de diversificación de tamaños del cuerpo y de especies. Aunque esto subraya la gran capacidad que tienen los entornos insulares en promover diversificación, diferentes grupos diversificando en las mismas islas no necesariamente siguen caminos evolutivos equivalentes. En el archipiélago de Socotra, la diversificación intra-isla en los géneros Hemidactylus y Haemodracon estuvo acompañada de grandes cambios del tamaño del cuerpo, que sólo posteriormente derivaron en cambios macroecológicos. Por el contrario, en el género Pristurus, la diversificación intra-isla fue principalmente mediada por cambios macroecológicos, siendo la diversificación del tamaño del cuerpo casi inapreciable. Esto demuestra que grupos cercanos diversificando en las mismas islas, pueden presentar patrones de diversificación completamente dispares. Al margen de las diversificaciones intra-isla expuestas, los resultados de esta tesis también han expuesto importantes patrones de diversificación también en el continente. Uno de ellos es el encontrado en los gecos pigopódidos australasiáticos, que después de adquirir una innovación clave (un fenotipo en forma de serpiente), entraron en dinámicas de diversificación comparables a los grupos insulares. Otro ejemplo destacado trabajado en esta tesis la diversidad encontrada dentro de la subespecie de geco Pristurus rupestris rupestris. Esta subespecie, en realidad, está formada por 14 especies altamente divergentes que diversificaron en las montañas del noroeste de Arábico, constituyendo uno de los casos más extremos de diversificación en vertebrados en Arabia.

El presente trabajo está centrado en la síntesis enantioselectiva de la cadena polihidroxílica presente en los productos marinos oscillariolida y formidolidas. Estas moléculas se pueden dividir en tres fragmentos; la cadena polihidroxílica, el macrociclo y el ácido graso. El presente trabajo está centrado en la síntesis enantioselectiva de la cadena polihidroxílica y el estudio en la unión del poliol y el macrociclo. La parte central del poliol se construye utilizando diferentes estrategias basadas en adiciones aldólicas. En la primera etapa se estudian auxiliares quirales como oxazolidinonas. El mejor resultado se observa con la (R)-4­benciloxazolidinona consiguiendo el aldol deseado con un exceso diatereomérico excelente y con buenos rendimientos. En la siguiente adición aldólica la inducción asimétrica es dirigida por los sustratos y se elabora un estudio para conseguir los aldoles de interés con excesos diasteroméricos y rendimientos satisfactorios. Se realizan adiciones aldólicas tipo acetato utilizando diferentes ácidos de Lewis como derivados de B o Ti, se aplica la variación de la las adiciones aldólicas tipo Mukaiyama y se prueba la adición de ácidos de Lewis quirales. Los mejores resultados se obtuvieron con el ácido de Lewis quiral DIP (bis(isopinocanfenil)cloroborano) rindiendo el aldol deseado con un excelente exceso diasteomérico y rendimientos moderados. La configuración absoluta del nuevo estereocentro se realiza utilizando el modelo de Mosher. A continuación, se lleva a cabo la reducción estereoselectiva de ß­hidroxicetonas a un compuesto dihidroxílico utilizando el catecolborono y se llega al intermedio 1,3-sin dihidroxílico con rendimientos y excesos diatereoméricos excelentes. De esta manera se sintetiza el fragmento central de la cadena polihidroxílica. En todo el proceso sintético cabe destacar la importancia de la ortogonalidad de los grupos protectores ya que es imprescindible la buena planificación para las desprotecciones selectivas y compatibilidad con las reacciones futuras. Partiendo del fragmento central de la cadena polihidroxílica, en un extremo se adiciona un derivado de la acroleína mediante la reacción de Barbier para conseguir un intermedio precursor al bromoéter de enol derivado. En el otro extremo, se estudia la adición de derivados organometálicos sobre aldehídos para poder dar como finalizada la síntesis total de la cadena polihidroxílica. Adicionalmente, se ha estudiado la unión del poliol con el macrociclo utilizando substratos modelo. La investigación se basa en la adición de un vinil metal derivado a un formil THF derivado. Se estudian tres metodologías diferentes: control por quelación con metales, carboaluminación y Nozaki­Hiyama-Kishi (NHK). En el estudio se consiguen identificar las mejores condiciones para obtener el producto de la adición con buenos rendimientos y excesos diastereoméricos. En todo este proceso sintético han sido imprescindibles técnicas como RMN (1D y 2D), HPLC, MS e IR. / There are an important number of marine origin molecules with promising bioactivity. The group of polyketide macrolides is an interesting family of molecules where oscillariolide and phormidolide are included. These two molecules contain a common polyhydroxy chain linked to a THF ring. First, oscillariolide was isolated from Oscillatoria sp. which allowed the structural elucidation of the polyhydroxy chain. After that, phormidolide was isolated and that permit the determination of the absolute configuration of the eight polyol stereocenters, which was not achieved until that moment. Although the structure of the polyol has been known during several years, the molecule has not been synthesized yet. The aim of this work is the asymmetric synthesis of the chain. Our target molecules can be separated in three different fragments: a polyhydroxy chain, a macrocycle and a fatty acid. One of the objectives of this work is the synthesis of the polyhydroxy chain and the study of the bond formation between the polyol and the macrocycle. Firstly, aldol condensations using different chiral auxiliaries were studied. Evans auxiliaries such as oxazolidinones and oxazolinthiones were studied. The reactions were performed using different chelating metals, bases, aldehyde protecting groups, and with different reaction times and temperatures. On the other hand, a metal based aldol condensation was carried out between a chiral aldehyde and a ketone. An important influence of the protecting group in the ß-position of the aldehyde on the stereochemical outcome of this transformation is suspected. The elongation of the chain was achieved using strategies bases on addition of organometallic reagents, as Barbier reaction and Reformatsky type reaction. Finally, the bond formation between the polihidroxy chain and the macrocycle of the natural products was studied. This study was carried out using model molecules. Three main methodologies were applied to study the vinylorganometilic reagents additions to chiral 2-formylTHF: chelated controlled organometallic addition, carboalumination and Nozaki-Hiyama-Kishi (NHK) reaction.

Research made with the collaboration of the Department of Microbiology, Faculty of Biology, and D D’ENGINY BIOREM S.L., / Nitrate pollution is a widespread problem that affects water bodies in many regions of the world, undermining water quality and therefore its safe use. Despite the application of improved management practices, nitrate pollution seems to increase, particularly in groundwater. The Nitrate Vulnerable Zone (NVZ) designation in Europe, for instance, has increased from 35.5% of the EU-15 territory at the end of 1999 to 44% at the end of 2003, and the Commission’s report for the period 2004-2007 revealed that 15% of groundwater monitoring stations in the EU-27 territory showed nitrate levels above the limit of 50 mg of nitrates per liter. Some trends towards nitrate attenuation are observed, but at least 33% of water bodies will clearly fail in achieving the 2015 goals set by the Water Framework Directive. Several efforts have been addressed to either reduce nitrogen inputs or to decrease its already accumulated levels, particularly by designing nitrate-removal technologies aimed at recovering drinking-water standards. This PhD thesis, hence, focuses on the optimization of an already existing technology for nitrate­removal: enhanced in situ biodenitrification (EISB), which is now regaining attention due to its economic and environmental benefits and its potential for scale-up and design of case­specific solutions. EISB is an engineered application of microbial heterotrophic denitrification aimed at in situ nitrate removal from groundwater. Aimed at stimulating facultative denitrifiers, EISB is based on the injection of a C source into the aquifer. Microbial denitrification is then enhanced in a designated area of the aquifer, creating a biologically active zone (often referred as biowall) which removes nitrate from the naturally-flowing groundwater. Among the different factors that affect the technical feasibility of EISB, the type and quantity of the injected C source is a key issue, particularly due to its influence upon the microbial processes that determine the treatment performance. The understanding of the subsurface geology and hydrogeology is also an issue of concern, particularly if highly heterogeneous media, such as fractured aquifers, are meant to be remediated. Aimed at achieving our research goal, several EISB experiments were developed at different scales -batch, flow-through column and pilot scale-and involving different geological media -granular and fractured-. Combined chemical, microbial and isotope monitoring tools where applied to gain a better insight on the denitrification process and thus improve technology design and optimization. The first set of batch-scale experiments focused on testing the viability of in situ heterotrophic denitrification and determining the most suitable biostimulants for a case­specific scenario in the Osona region, a Catalan NVZ showing historic nitrate pollution up to 200 mg/L. Native microbiota was stimulated and nitrate reduction was effectively achieved by addition of a carbon source (ethanol or glucose) as well as a phosphorous source (disodium hydrogen phosphate). Transient nitrite accumulation was observed, especially when using glucose as the C source. The N and O isotope fractionation was determined to be -13.0‰ and -17.1‰ for eN and -8.9‰ and -15.1‰ for eO in ethanol and glucose-amended experiments respectively, resulting in eN/eO values of 1.46 (ethanol-amended experiment), and 1.13 (glucose-amended). Organic carbon (OC) consumption in batch­scale experiments, expressed as .C/.NO3 -, varied slightly depending on the type of C source used: 1.6 mmolOC/mmolNO3 -for ethanol and 2.2 for the glucose, similarly to stoichiometric values associated with nitrate respiration (0.83 and 1.25 mmolOC/mmolNO3 ­respectively). When deriving stoichiometric reactions that accounted not only for the amount of electron donor used for nitrate respiration but also for cell synthesis, the following values were determined: 1.9 and 2.0 mmolOC/mmolNO3 -for ethanol and glucose­induced biodenitrification respectively. These values were used for the numerical modeling of batch-scale experiments, aimed at quantifying microbial kinetics by applying the modified Monod expression. The (geochemical) numerical model also indicated a different effect of mineral precipitation on ethanol or glucose-induced denitrification, an effect that is linked to a different alkalinity production. Such effect could be taken into account when designing and/or optimizing EISB systems, particularly as a way to control geochemical clogging. A pilot-scale application was then performed at the site, aimed at assessing the viability of EISB in a fractured aquifer. Ethanol was now used as the main C source, and based on lab­scale results, P was also added. Again, transient nitrite accumulation was detected, and evidences for incomplete denitrification and coexistence of other respiration processes (such as iron or sulfate reduction) and autotrophic denitrification were observed. Sulfate isotope characterization proved that autotrophic denitrification linked to sulfide oxidation could be occurring along with heterotrophic denitrification, while sulfate­reduction couldn’t be verified. Overall, results suggested that stimulated heterotrophic denitrification could be applied as a remedial alternative in a fractured media and despite the complexity of the formation. However, a deep understanding of the system is required and efforts must be addressed to control microbial population and stability as a key issue to avoid the decrease of groundwater quality due to incomplete denitrification or secondary respiratory processes. Different engineering approaches such as feeding or pumping strategies could help improving the system performance. Aimed at testing the impact of such engineering approaches upon resulting water quality, a second study-case was studied, now in an alluvial media. . A flow-through experiment was built to simulate an EISB system and assess the influence of different C addition strategies upon the denitrification process. Heterotrophic denitrification was stimulated by the periodic addition of a C source (ethanol), and 4 different addition strategies were evaluated, being the first-one a weekly injection, and the others a daily injection with decreasing amounts of C. Enhanced denitrification was stimulated following the first C addition, easily achieving drinking water standards for both nitrate and nitrite. Water quality in terms of remaining C, denitrification intermediates and other anaerobic respiration products varied during the experimental time. Ethanol, for instance, showed a cyclic behavior during the weekly feeding strategy while it was completely depleted when injected daily. A quasi steady­state nitrate outflow, similar to ethanol’s, was obtained in daily injection scenarios, with nitrate levels ranging from non-detected values and up to 10 mg/L, and nitrite’s remaining undetected. No dissimilatory nitrate reduction to ammonium was ever detected and some secondary microbial respiration processes, mainly manganese reduction, were suspected to occur temporarily. Overall, results showed that biodenitrification could be successfully achieved by a daily addition of a C source slightly higher than the stoichiometric value, diminishing the accumulation of non-desired products and the biofilm growth and still obtaining the required denitrification results. Reducing the C/N ratio enables us to reduce treatment costs while achieving a better water quality in terms of remaining C and residual microflora, and potentially reducing the biofouling effect due to the increase of endogenous respiration. Endogenous activity –that provides internal C for denitrification-may become important when low C/N values are used, keep denitrification temporarily ongoing and reducing the biofilm growth, but may affect the biodenitrification performance at longer operation times. Such aspects should be further evaluated using modeling and/or experimental tools. Furthermore, results suggested that not only the feeding strategy but also the biofilm life-time have a direct effect on microbial population structure and hence on the biodenitrification performance, reducing the accumulation of nitrite over time. The obtained eN/eO fractionation values for the flow-through experiment (1.01) fell within the low-end of previously reported data (varying from 0.9 to 2.3), an effect that may be linked to faster microbial kinetics in enhanced vs. natural biodenitrification. Similar low values were observed in our previous batch-scale experiments as well as in other work conducted in our lab. Concerning ethanol’s fractionation, on the other side, a two-trend behavior was observed, probably indicating a change in the dominating C­consuming population. Interestingly, the second trend suggests an inverse fractionation of the C source that got depleted while being consumed. / Esta tesis se destina a la evaluación de la viabilidad técnica de la biodesnitrificación in situ de aguas subterráneas contaminadas por nitratos, con el objetivo de optimizar las estrategias de bioestimulación y mejorar los resultados del proceso microbiano. El proyecto evalúa la aplicación de la tecnología en dos entornos geológicos distintos: un medio fracturado de baja porosidad y un aluvial arenoso. Se desarrollan ensayos a tres escalas distintas: batch, columna de laboratorio de flujo continuo y piloto. El seguimiento y estudio del proceso se desarrolla mediante la combinación de herramientas de análisis químico y microbiológico y la aplicación de isótopos estables del nitrato, el sulfato y el C.

Tesi realitzada a l'Institut de Química Avançada de Catalunya (IQAC-CSIC) / Oxidative stress is one of the most important factors in degenerative diseases like cancer, Alzheimer or in episodes like ischemia. During these processes, reactive species of oxygen (ROS) and nitrogen (RNS) are generated. This fact can cause the chemical modification of important biomolecules, in particular lipids and proteins. The strategy that involves the use of antioxidants and neuroprotective agents to fight against the lesive effects of these species stumbles over the blood-brain-barrier (BBB). This barrier protects the brain from the action of a wide variety of organic molecules and drugs. Thus, it is important to develop novel antioxidant agents with good delivery through the BBB. In our group it was discovered the antioxidant agent 3,4-dihydro-2,2-dimethyl-7-metoxy-1-(2H)-benzopyran (CR-6), an analogue of alpha- and gamma-tocopherols (Figure 1), which is currently used in dermopharmacy and it is also in a phase II trials for anticancer treatment (combined with other drugs). In our present project, one of the main goals is the synthesis of a short library of CR-6 analogues that could improve the pass through the BBB. Accordingly, fourteen novel CR-6 analogues have been synthesized introducing essential nutrients for brain that work as BBB-shuttles at C2 of CR-6 scaffold. The antioxidant activity was evaluated to assure that it does not change by the incorporation of variability at C2 position (the DPPH assay and the in vitro CAA were used). In addition, the BBB permeability was evaluated to compare the BBB bioavailability of these new antioxidant agents with the references CR-6 and Trolox (PAMPA, Caco-2 and BBCEC assays were used). / El estrés oxidativo es uno de los factores etiológicos más importantes en las enfermedades degenerativas como el cáncer, el Alzheimer o en episodios de isquemia. Durante estos procesos, se generan especies reactivas de oxigeno (ROS) y de nitrógeno (RNS) que provocan modificaciones de las biomoléculas como los lípidos, las proteínas y el ADN. El uso de agentes antioxidantes y neuroprotectores ayudan a reducir los efectos lesivos que el estrés oxidativo tiene en el cerebro, por ejemplo. La barrera hematoencefálica o BBB protege al cerebro de la acción de una amplia variedad de moléculas orgánicas y fármacos. Por ello, existe un gran interés en el desarrollo de nuevos agentes antioxidantes con un buen transporte a través de la BBB. En nuestro grupo de investigación se ha desarrollado el agente antioxidante 3,4-dihidro-2,2-dimetil-7-metoxi-1-(2H)-benzopirano (CR-6), un análogo de alfa- y gamma-tocoferol, que en la actualidad se emplea en dermofarmacia y se encuentra en ensayos de fase II para el tratamiento del cáncer (combinado con otros fármacos). En este proyecto se persigue la síntesis de una colección de análogos del CR-6 que mejoren el paso a través de la BBB. De este modo, se han sintetizado catorce compuestos derivados del CR-6 introduciendo nutrientes esenciales del cerebro que actúan como transbordadores de la BBB. La actividad antioxidante de todos estos compuestos se ha evaluado para asegurar que ésta se mantiene al introducir variabilidad en la posición C2 mediante los ensayos del DPPH y el in vitro CAA. A su vez, la permeabilidad de la BBB se ha evaluado para compararla con los compuestos de referencia CR-6 y Trolox a partir de los ensayos in vitro Caco-2, PAMPA y BBCEC.

The aim of this thesis was to study quantum phenomena and excitation modes in type-I superconductors and magnetic vortices. The intermediate state in type-I superconductors is characterized by the gradual penetration of magnetic flux and the coexistence of normal and superconducting domains. This thermodynamic phase shows a magnetic irreversibility of topological origin, even in the case of defect-free samples. This irreversibility has been explored in disk-shaped samples made of lead (the prototype of a type-I superconductor) using a magnetic field applied perpendicularly to the disk plane, by means of the measurement of hysteresis cycles at different temperatures, zero-field-cooled and field-cooled magnetization curves at different magnetic fields and magnetic relaxations along the descending branch of the hysteresis cycles. Non-thermal magnetic relaxations have been observed in these samples at low temperatures, which have been attributed to the tunnel effect of normal-superconductor interfaces through pinning energy barriers. A quantum model based on the Caldeira-Leggett theory for dissipative systems have been developed to explain these experimental observations. The interface is described as a 2D elastic manifold that is pinned by a planar defect. The pinning barrier can be controlled by a supercurrent that exerts a force on the interface. The vortex state turns out to be the ground state of magnetic disks for a wide variety of thicknesses and radii. It is characterized by the curling of the magnetization in the plane of the disk, leaving virtually no magnetic ‘charges’. The very weak uncompensated magnetic moment of the disk sticks out of a small area confined to the vortex core. The low-frequency dynamics of the vortex state is characterized by the spiral-like precessional motion of the vortex core as a whole (gyrotropic mode), which can be induced by the application of an in-plane magnetic field. The presence of structural defects in these magnetic disks affects the dynamics of the vortex state, which is indicative of the elastic nature of the vortex core along the axial direction of the disk. It has been studied whether the gyrotropic mode allows spatial dispersion similar to spin waves of a finite wavelength in ferromagnets. The excitation spectrum splits into two branches, one related to the gyrotropic mode with a gap given by the gyrofrequency of the disk and the other related to the existence of an effective mass associated to the vortex core. The magnetic irreversibility of the vortex state has been also explored by means of an experimental protocol analogous to that used in the case of type-I superconductors. Non-thermal magnetic relaxations have been observed again at low temperatures, which is attributed to the tunnel effect of a segment of the vortex core line through pinning barriers. A quantum model based on the Caldeira-Leggett theory for dissipative systems have been developed to explain these experimental observations. The interface is described as a 1D elastic manifold that is pinned by a linear defect. To conclude, the effect of the vortex state on the supercurrent of a Josephson junction has been studied in the case where the non-superconducting layer consists of a magnetic disk with the vortex as the ground state. It has been concluded that the variation of the Josephson current with tiny displacements of the vortex core can be detected experimentally. / El objetivo de esta tesis ha sido estudiar fenómenos cuánticos y modos de excitación en superconductores tipo-I y vórtices magnéticos. La irreversibilidad magnética en muestras de plomo con forma de disco en el estado intermedio ha sido explorada mediante medidas de ciclos de histéresis a diferentes temperaturas, medidas de las curvas de magnetización zero-field-cooled y field-cooled a diferentes campos y relajaciones magnéticas a lo largo de la rama descendiente de los ciclos de histéresis. Se han observado relajaciones magnéticas independientes de la temperatura en estas muestras, las cuales se atribuyen al efecto túnel de las interficies normal-superconductor a través de barreras de anclaje. Un modelo de efecto túnel basado en la teoría de Caldeira-Leggett para sistemas disipativos se ha construido para explicar estas observaciones experimentales, donde la interfície se trata como una variedad 2D elástica que se ancla a defectos planares. La barrera de anclaje se puede controlar mediante la inyección de supercorriente en el sistema. El núcleo del estado vórtice muestra una naturaleza elástica a lo largo de la dirección axial de los discos magnéticos que lo presentan como estado fundamental. Se ha estudiado bajo qué condiciones el modo girótropo es compatible con una dispersión espacial semejante a las ondas de espín de longitud de onda finita presentes en un ferromagneto. El espectro de excitaciones axiales presenta dos ramas bien definidas, una asociada al modo girótropo y la otra originada por la existencia de una masa efectiva asociada al núcleo. También se ha explorado la irreversibilidad magnética del estado vórtice mediante un protocolo análogo al de los superconductores tipo-I. De nuevo se ha observado un comportamiento no térmico a bajas temperaturas en las relajaciones magnéticas, el cual es atribuido al efecto túnel de un segmento del núcleo vorticial a través de las barreras de anclaje. Un modelo de efecto túnel basado en la teoría de Caldeira-Leggett para sistemas disipativos se ha construido para explicar estas observaciones experimentales, donde el núcleo vorticial se trata como una variedad 1D elástica anclada a un defecto lineal. Por último, se ha estudiado cuál sería el efecto del estado vórtice sobre la supercorriente de una unión Josephson si como capa no superconductora se escogiera un disco magnético con este estado fundamental. Se ha concluido que la variación de la corriente Josephson con desplazamientos pequeños del núcleo vorticial es detectable experimentalmente.

Tesi realitzada a l'Institut de Recerca Biomèdica de Barcelona (IRBB) / Prolyl oligopeptidase (POP) is an 81-KDa bidomain enzyme which hydrolyses short proline-containing peptides. This enzyme is involved in mnemonic and cognitive processes, and the dysregulation of POP activity is related to mental diseases. Probably, POP modulates the phosphoinositide signalling pathway through protein-protein interactions (PPI). Hence, the development of POP inhibitors is an area of great interest for the treatment of cognitive deficits associated with mental and neurodegenerative diseases. Recently, it has been found that the administration of POP inhibitors increases the clearance of a-synuclein aggregates in vivo, indicating that POP can be related to some extend with the pathogenic conditions of Parkinson’s disease (PD). Probably, this increase in the a-synuclein catabolism might be a consequence of a direct interaction between the two proteins. For this reason, POP inhibitors might be drug candidates for the preventive treatment of PD. Although the X-ray structure of POP is well studied, it is not clear which are the conformational fluctuations responsible for the circulation of substrates and products during the catalytic cycle. Several studies suggest that loops surrounding the active site are involved in a gating mechanism, while others postulate that interdomain separation might expose the active site. Moreover, such conformational transitions might be essential for the recognition events of POP. The elucidation of the conformational landscape of POP is a challenging task due to the high molecular weight of the enzyme. In this PhD thesis we have used a combination of robust biophysical tools (in particular, NMR and SAXS) together with molecular dynamics simulations (MD) in order to decipher the conformational dynamics of POP in solution. In addition, POP was also analysed by ion mobility mass spectrometry, an emerging biophysical tool in structural biology. Finally, we performed preliminary studies of the interaction between POP and a-synuclein by NMR. The results obtained in this PhD thesis demonstrated that POP exists in solution in a slow conformational exchange between open and closed conformations. The conformational transitions involved the periodic separation of the two domains in a hinge-type motion. Relaxation dispersion experiments showed that this long range conformational transition was better described by several independent motions of different amplitudes, stressing the highly dynamic behaviour of POP. Moreover, the analysis of SAXS data complemented by MD simulations found that the interdomain separation caused the inactive arrangement of the active site. This suggests that the separation between domains might be critical for substrate recruitment and product release. Of interest, inhibitors caused the total displacement of this equilibrium towards the stabilized closed conformation, therefore quenching dynamics and the catalytic activity. The study of the interaction between POP and a-synuclein by NMR disclosed that both proteins might be involved in a weak and transient interaction. Of interest, this interaction showed more affinity in the case of POP bound to inhibitors. In this case, interaction specially affected a broad segment of the C-terminal region of a-synuclein. This result suggested that the recognition between the two proteins depends on the conformational state of POP. Therefore, modulating the conformational landscape of POP by inhibitors might control this interaction. In summary, the results obtained in this PhD thesis demonstrated that POP undergo slow exchange between open and closed conformations in solution, and found that inhibitors have deep effects in the native conformational landscape of POP. Of interest, these conformational transitions might be essential for regulating the PPI necessary for the biological function of POP. Hence, the in vivo effects of POP inhibitors might result as a consequence of the alterations in the recognition events of POP. / La prolil oligopeptidasa (POP) es un enzim de 81 KDa que hidrolitza pèptids curts amb contingut en prolina. La POP actua en el sistema nerviós central mitjançant interaccions proteïna-proteïna (IPP), i la seva funció biològica està relacionada amb la memòria i els processos cognitius. Per aquesta raó, els inhibidors de la POP són compostos d’interès terapèutic per al tractament dels dèficits cognitius. Recentment, s’ha descobert que els inhibidors de la POP poden prevenir la patogènesis de la malaltia de Pàrkinson, probablement a través d’una interacció directa entre la POP i l’a-sinucleïna (la principal proteïna causant dels processos neurodegeneratius de la malaltia de Parkinson). Tot i que l’estructura cristal·logràfica de la POP està ben definida, no es sap quines són les transicions conformacionals que permeten completar el cicle catalític de la POP. Probablement, aquesta riquesa conformacional també té un paper rellevant en el control de les IPP. Malauradament, l’estudi conformacional complet de la POP és tot un repte degut al seu elevat pes molecular. En aquesta tesis doctoral s’ha emprat una combinació de tècniques biofísiques avançades (en concret, la resonància magnètica nuclear, la dispersió de raigs X de baix angle, i l’espectrometria de masses de mobilitat iònica) conjuntament amb simulacions de dinàmica molecular, per tal d’analitzar la dinàmica conformacional de la POP en solució. A més, s’ha estudiat la possible interacció entre la POP i l’a-sinucleïna mitjançant experiments de RMN. Els resultats obtinguts en aquesta tesi doctoral han demostrat que la POP es troba en solució en un equilibri conformacional lent entre conformacions obertes i tancades, originades a partir de la separació entre dos dominis. Els inhibidors de la POP causen una gran estabilització de la conformació tancada, amb la qual cosa l’equilibri dinàmic es desplaça totalment cap a aquesta conformació. A més, es va poder detectar una interacció dèbil i transitòria entre la POP i l’a-sinucleïna, que esdevenia especialment afavorida en la presència d’inhibidors. Així, els nostres resultats suggereixen que la diversitat conformacional de la POP es necessària per a la seva funció, i que els inhibidors poden desencadenar la seva funció biològica desplaçant l’equilibri conformacional.

1. Tendència relativa a formar enamines de diferents aldehids i cetones S’ha treballat en l’estudi per RMN de 1H de diverses espècies que estan implicades en reaccions organocatalítiques. L’equilibri entre compostos carbonílics i les seves enamines ha estat estudiat en DMSO-d6. La utilització del (S)-prolinol protegit en forma de TBDPS (1) ens ha permès comparar diferents substrats. S’ha establert una escala sobre la tendència de aldehids i cetones a formar enamines per mitjà de reaccions d’intercanvi per parelles. Les constants d’equilibri van ser determinades per la integració dels senyals corresponents per RMN de 1H. Les enamines més estables són aquelles que estan conjugades amb un anell aromàtic. Les enamines de les cetones tipus 1,3-dioxan-5-ona són sorprenentment estables. Les cetones alifàtiques mostren una baixa tendència a formar enamines. 2. Oxazolidinones i enamines de L-prolina Hem estudiat la capacitat de la prolina (L-Pro) per formar enamines i/o oxazolidinones en DMSO-d6. Els aldehids a-O-substituïts amb L-Pro només donen oxazolidinones, mentre que els aldehids lineals donen enamines i oxazolidinones i les enamines predominen quan el grup enamino està conjugat (amb un anell aromàtic, per exemple). S’ha demostrat que els aldehids quirals a-sililoxi i a-alcoxi derivats tenen una gran tendència a formar oxazolidinona (exo) amb prolina, reduint la capacitat de formació d’enamina i evitant la racemització del centre en a. S’ha demostrat, també, que la 2,2-dimetil-1,3-dioxan-5-ona, tan emprada en reaccions aldòliques catalitzades per prolina, té una gran tendència a formar enamina, a diferència d’altres cetones cícliques. 3. Reaccions aldòliques de 2,2-dimetil-1,3-dioxan-5-ona i aldehids a-substituïts catalitzades per prolina Degut al fet que la 2,2-dimetil-1,3-dioxan-5-ona (dioxanona 12) és una cetona cíclica àmpliament utilitzada en organocatàlisi com a nucleòfil (via la seva enamina) en reaccions catalitzades per prolina i tenint en compte el nostre treball sobre formació d’oxazolidinones i/o enamines amb prolina, s’ha estudiat la reacció aldòlica catalitzada per prolina de 12 amb diferents aldehids a-substituïts La reacció aldòlica no té conversions totals en absència d’aigua tal com s’observa en la reacció monitoritzada per RMN de 1H, essent entre 5 i 25 equivalents d’aigua els necessaris per obtenir conversions totals. L’obtenció de l’aldol com a únic diastereòmer de la reacció posa de manifest que el mètode és potent per a la preparació de poliols. Això és degut, en part, a la formació de l’oxazolidinona de l’aldehid sense passar per la seva enamina. 4. Síntesi total de la (–)-amfidinolida K Hem sintetitzat la ()-amfidinolida K segons la nostra retrosíntesi que implica la desconnexió de la (–)-amfidinolida K (40, C8–C9 i C1–O) en dos fragments, sud-oest (o sud-occidental, 41) i nord-est (o nord-oriental, 42), tal com s’indica a la figura 8. La unió dels dos fragments s’ha dut a terme via reacció d’al·lilació d’Hosomi–Sakurai emprant un aciloxiborà quiral (CAB) per induir l’estereoselectivitat a la reacció. Una reacció de macrolactonització de Yamaguchi o de Shiina tancaria la molècula tot donant lloc a l’anell de 19 membres. El fragment sud-oest, al mateix temps, prové d’una reacció de Negishi entre un bromoalquè comercial (43) i un iodoalquè (44), mentre que el fragment nord-est prové de la reacció aldòlica entre el producte acilat d’Evans 45 i l’aldehid 46. S’ha dut a terme l’optimització de desprotecció de l’alcohol primari del compost 88 amb TBAF amb èxit. Finalment, una macrolactonització en condicions de Shiina i posterior epoxidació de Sharpless ha donat lloc a la síntesi total de la (–)-amfidinolida K. En experiments in vitro amb actina, la (–)-amfidinolida K ha mostrat un fort efecte estabilitzant de la F-actina (aproximadament un 70% respecte a la fal·loïdina). / 1. Relative tendency of carbonyl compounds to form enamines Equilibria between carbonyl compounds and their enamines have been examined by NMR spectroscopy in DMSO-d6. Use of O-TBDPS-protected prolinol (1) as amino-catalyst has allowed us to compare many substrates. A general scale of the tendency of carbonyl compounds to form enamines has been established by means of the exchange reaction between pairs. Equilibria constants were easily determined from the integration of the corresponding 1H NMR peaks. The most stable enamines are those conjugated with an aromatic ring (provided that there is no steric inhibition of the resonance). 1,3-Dioxan-5-one enamines are surprisingly stable. Aliphatic ketones show a very low trend to form enamines. 2. Oxazolidinone/enamine ratios in the reactions of aldehydes and ketones with proline The trend of (S)-Pro (Pro) and carbonyl compounds to form enamines or oxazolidinones in DMSO-d6 has also been examined (Fig 4). a-Substituted aldehydes and Pro have a tendency to afford oxazolidinones rather than enamines, preventing the racemization of a-stereocenters. The oxazolidinone/enamine ratios has been examined in the reaction of Pro with aldehydes and ketones. 3. Proline-catalyzed aldol reaction of 2,2-dimethyl-1,3-dioxan-5-one with a-substituted aldehydes We have examined the aldol reaction of 2,2-dimethyl-1,3-dioxan-5-one with (S)-2-(tert-butyldiphenylsilyloxy)propanal, (S)-2-(tert-butyldimethylsilyloxy)propanal and (S)-2-(4-methoxybenzyloxy)propanal using Pro as the catalyst. The addition of water (5-25 equiv.) plays an important role in order to release the aldehyde from the oxazolidinone species. Complete conversions have been achieved when the aldol reaction was carried out during 40 h in presence of water. 4. Total synthesis of (–)-amphidinolide K (–)-Amphidinolide K is a cytotoxic macrolide that was isolated from a marine Amphidinium sp. dinoflagellate by Kobayashi et al. Williams and Meyer, who achieved the syntheses of several stereoisomers, including the enantiomer of amphidinolide K, concluded that the absolute configuration of the natural product is 40. A total synthesis of 40 has been recently reported by Lee et al. We have reported now a second total synthesis of the natural enantiomer. Our synthesis of (.)-amphidinolide K is based on the asymmetric allylation of enal C9-C22 with allylsilane C1-C8. The final macrolactonization was achieved by the Shiina method in good yield. The synthesis of fragment C1-C8 was attained by Negishi coupling. The required alkenyl iodide was prepared by in situ hydrozirconation-iodination from the corresponding alkyne. The key step in the formation of fragment C9-C22 is the indicated Evans aldol reaction.

La presencia de contaminantes y residuos químicos en los alimentos es un problema que actualmente genera gran preocupación, ya que presenta importantes implicaciones para la salud y la seguridad pública. En este sentido, el uso indiscriminado, inadecuado y/o fraudulento de fármacos veterinarios puede dar lugar a la presencia de residuos de los mismos en los alimentos de origen animal, entrando de esta forma en la cadena trófica y ocasionando riesgos para la salud. Por otro lado, este tipo de residuos puede ser liberado al medioambiente directamente o indirectamente a través de la utilización del estiércol procedente de animales tratados, generando de esta manera la contaminación de suelos y aguas. En el caso de los antibióticos, la presencia de cantidades residuales en alimentos y en el medioambiente ha sido identificada como una de las causas de la aparición de mecanismos de resistencia a los antimicrobianos por parte de patógenos que causan enfermedades humanas, lo que es motivo de gran preocupación por parte de las autoridades sanitarias, diferentes agencias y organizaciones gubernamentales y de la sociedad en general. Por lo tanto, existe la necesidad de garantizar la seguridad y la calidad de los productos alimenticios, tal es así que los consumidores son cada vez más exigentes, obligando a la industria alimenticia a tener en cuenta sus preocupaciones, en términos de una producción de alimentos más naturales, ecológicos y saludables con un origen claramente rastreable de todos sus ingredientes. En esta tesis se describe la investigación realizada respecto al desarrollo de técnicas de análisis alternativas que permitan incrementar la eficiencia del control de residuos de antibióticos en alimentos, en base a la utilización de receptores selectivos (anticuerpos). Concretamente, el objetivo final de esta tesis se enfocó al desarrollo de técnicas inmunoquímicas e inmunosensores para la detección de residuos de antibióticos de tipo quinolona en leche. En este sentido, la producción de anticuerpos con selectividad de clase para quinolonas, una de las familias de antibióticos más importante en el ámbito veterinario, ha sido uno de los objetivos principales de este trabajo de investigación. De esta manera, se ha abordado la producción de dichos anticuerpos mediante el diseño racional y síntesis de los haptenos apropiados, utilizando el criterio químico y herramientas de modelización molecular. La evaluación de las características de los anticuerpos generados se ha realizado a través del desarrollo de ensayos de tipo ELISA en microplaca. Los resultados muestran que ha sido posible obtener anticuerpos de clase selectivos para quinolonas, ya que pueden ser detectados hasta diez congéneres de dicha familia con una detectabilidad adecuada para los requerimientos de la Comisión Europea. A su vez, se han desarrollado los protocolos necesarios para la aplicación de estos procedimientos inmunoquímicos al análisis de muestras de leche, estableciéndose un formato semicuantitativo capaz de identificar claramente las muestras libres de quinolona de las contaminadas. Asimismo, los inmunoreactivos y procedimientos inmunoquímicos establecidos se han implementado en un dispositivo inmunosensor amperométrico, basado en la utilización de partículas magnéticas. En este caso, a pesar de la complejidad de la leche, el uso de partículas magnéticas ha permitido la eliminación de las posibles interferencias causadas por los componentes de la matriz, pudiéndose realizar mediciones cuantitativas directamente en muestras de leche, sin ningún tratamiento de muestra adicional o etapa de extracción. Dicho inmunosensor es capaz de detectar hasta siete quinolonas diferentes por debajo de los límites máximos de residuos (LMRs) definidos por el Comisión Europea para la leche, ofreciendo una estrategia prometedora para el análisis in situ, rápido, simple y de bajo costo, de antibióticos de tipo quinolona en muestras complejas. / In this thesis, research on the development of alternative analysis techniques increasing the efficiency of the control of antibiotic residues in food is described based on the use of selective (antibodies) receptors. Particulary, the final objective of this thesis was focused to the development of immunochemical techniques and immunosensors for detection of quinolone antibiotic residues in milk. In this sense, the production of class selectivity antibodies for quinolones, one of the most important families in the veterinary antibiotics, has been one of the main objectives of this research. The production of antibodies has been addressed by rational design and synthesis of suitable haptens, by means chemical criteria and molecular modeling tools. The evaluation of the characteristics of the antibodies produced was performed by developing microplate ELISA test. Results show that it has been possible to obtain class selective antibodies for quinolones, able to detected up to ten quinolones with suitable detectability for the requirements of the European Commission. Moreover, protocols for the implementation of these immunochemical methods to the analysis of milk samples have been developed, establishing a semiquantitative format able to clearly identify quinolone free samples from the contaminated. Also, the established immunochemical procedures and immunoreagents developed have been implemented in an amperometric immunosensor device based on the use of magnetic particles. In this case, despite the complexity of the milk, the use of magnetic particles has allowed the elimination of potential interference by matrix components, being able to make quantitative measurements directly in milk samples without any further sample treatment or extraction step. The immunosensor is able to detect up to seven different quinolones below the maximum residue limits (MRLs) established by the European Commission for milk, offering a promising strategy for the in situ, fast, simple and inexpensive analysis of quinolones in complex samples.

El grup prostètic hemo és un component essencial de moltes proteïnes, com l'hemoglobina, la mioglobina i els citocroms. Existeixen quatre reaccions concatenades en la síntesi del grup hemo que són comunes en els diferents regnes animal, vegetal, i bacterià. Dues d'aquestes reaccions estan catalitzades per l'enzim Porfobilinogen Desaminasa (PBGD) i l'enzim Uroporfirinogen III Sintasa (U3S). Alteracions en l'activitat enzimàtica de la U3S donen lloc a una malaltia, la Porfíria Eritropoiètica Congènita (PEC). En les últimes dècades s'han incrementat els anàlisis genètica a pacients, facilitant una gran quantitat d'informació que relaciona aquesta malaltia amb alteracions en la seqüència d'aminoàcids de l'enzim U3S. Per determinar l'activitat enzimàtica dels enzims mutants relacionats amb la patologia s'han clonat, expressat i purificat les 25 mutacions puntuals patogèniques trobades en pacients, així com les proteïnes U3S salvatge i PBGD (necessària per dur a terme l'assaig enzimàtic). Els resultats de l'activitat relativa d'aquests enzims mutants poden explicar un seguit de fenotips, però no la seva totalitat. De fet, l'anàlisi dels resultats de l'activitat enzimàtica ha revelat que la gran majoria de les mutacions retenen una gran quantitat de l'activitat catalítica de la forma salvatge.Per entendre l'efecte d'algunes mutacions, s'ha realitzat un detallat estudi estructural i termodinàmic d'aquestes proteïnes. Amb aquest objectiu, s'ha determinat l'estabilitat cinètica d'aquestes proteïnes mutants respecte a la proteïna salvatge, i s'ha observat que en alguns casos, la mutació afecta l'estabilitat de l'enzim. Aquests estudis han permès identificar una regió helicoïdal de la proteïna imprescindible per al manteniment de l'estructura nativa.Per altra banda, s'havia descrit que la mutació C73R es trobava present en més d'un terç dels pacients de PEC, sent una mutació associada als fenotips més greus. Amb la col·laboració del Dr. Juan Manuel Falcón i el seu equip, i amb l'objectiu d'entendre l'efecte d'aquesta mutació en els sistemes vius, s'han realitzat una sèrie d'experiments in vivo que han permès determinar l'estabilitat relativa de la mutació C73R en l'entorn cel·lular, essent aquesta molt inferior respecte a l'enzim salvatge i mostrant un patró de degradació intracel·lular prematur. Nombrosos estudis han posat de manifest la possibilitat de manipular el plegament proteic dins de la cèl·lula mitjançant la utilització de "xaperones químiques". S'ha vist que l'ús d'aquests compostos pot aplicar-se en varies malalties associades amb pèrdues de la funció degudes a mutacions desestabilitzants. Estudis preliminars amb "xaperones químiques" han permès retenir la proteïna mutant C73R intracel·lular evitant-ne la degradació, obrint així, una nova línia d'investigació terapèutica. / The term porphyria describes a family of autosomal diseases produced by the existence of one or more mutations in any of the enzymes in the heme group biosynthetic pathway. In particular, a deficient Uroporphyrinogen III Synthase (U3S) activity is ultimately responsible for the human Congenital Erythropoietic Porphyria (CEP). The severity of the disease is usually related to the residual enzyme activity. In an effort to better understand the relationship between U3S malfunction and CEP, we set out to obtain a detailed characterization of the 25 pathogenic missense mutations of U3S. Comparing the enzymatic activity results, we demonstrate that in some cases, enzyme activity is not the only explanation for the severity of the disease. Our hypothesis is that mutants can also alter protein unfolding thermodynamics, so stability properties of U3S have been investigated. We have demonstrated that protein unfold takes place so fast in a subset of mutations that retain most of the synthase activity, which explain their CEP phenotype. Furthermore, an important key helix for protein stability has been identified.Through collaboration with Dr. Juan Manuel Falcón, in vivo behaviour of the wild-type human U3S and the most frequently mutation found in CEP patients, C73R mutation, have been investigated. Preliminary results indicate that C73R mutant is expressed in some mammal cells, but its in vivo stability is lower than in case of the wild-type. Our recent experiments with chemical chaperones have increased C73R mutant in vivo stability, suggesting a new potential therapeutic treatment.

Les espècies d'aranyes del gènere Loxosceles són originàries de les zones temperades i tropicals d'Amèrica i d'Àfrica. La major part de les espècies tenen àrees de distribució relativament petites, tot i que existeixen algunes excepcions a aquest patró general, i algunes espècies amb hàbits antropòfils han estat transportades de forma passiva i introduïdes en diverses zones del planeta. Aquest és el cas, per exemple, de L. rufescens (Dufour 1820), l'única espècie descrita d'Europa. Actualment, la diversitat del gènere es concentra a Amèrica del Sud i del Nord, d'on s'han descrit la major part de les 107 espècies del gènere. L'objectiu principal d'aquesta tesi ha estat estudiar la diversitat del gènere Loxosceles a la Mediterrània, el Nord d'Àfrica i les Illes Canàries, i situar-la en un context filogenètic. L'intens mostreig de camp portat a terme per tota l'àrea d'estudi a posat al descobert l'existència d'una elevada diversitat. S'han identificat 11 llinatges mitocondrials dins de L. rufescens amb dos patrons filogeogràfics contrastats: (1) uns llinatges amb poblacions ben estructurades al Marroc i la Península Ibèrica, i (2) uns llinatges que manquen d'estructura geogràfica al llarg de la Mediterrània. Les estimes d'edats han situat la major part de diversificacions durant el Pleistocè, i la diferenciació al·lopàtrica dels llinatges és compatible amb els múltiples refugis Pleistocènics identificats amb la modelització de nínxol ecològics (ENM). Sembla que el transport indirecte pels humans ha complicat la biogeografia actual en aquesta espècie. En el cas de les Illes Canàries, les anàlisis filogenètiques han posat al descobert l'existència d'un clade ben recolzat endèmic de les Illes Canàries, format per set llinatges evolutius distribuïts de forma al·lopàtrica. La major part de dispersions entre Illes van succeir durant el Miocè superior. A més, també s'han trobat representants de l'espècie cosmopolita Loxosceles rufescens en l'arxipèlag. Sis dels llinatges pels quals es disposava de representants adults, s'han estudiat morfològicament i s'han descrit com a espècies nominals, quedant la diversitat de Loxosceles en les Illes Canàries amb una espècie endèmica de Fuerteventura i Lanzarote, dues de Gran Canària, dues més de Tenerife i una de La Gomera i El Hierro. Per tal d'aprofundir en la filogeografia de l'espècie endèmica de les illes i illots orientals, s'han desenvolupat set microsatèl·lits polimòrfics utilitzant les noves tecnologies de seqüenciació. Aquests nous marcadors han servit per a ressaltar la importància dels canvis en el nivell del mar i el vulcanisme en l'estructuració genètica d'aquesta espècie. Pel que fa a la diversitat de Loxosceles del Nord d'Àfrica, s'ha descrit una nova espècie de Loxosceles de Tunísia, L. mrazig, representant la segona espècie del gènere en la Conca Mediterrània. Tot i descriure's primerament d'una sola localitat, durant mostrejos posteriors s'han localitzat en diverses zones àrides del Nord d'Àfrica, des del Marroc fins a Israel. A més, l'estudi en profunditat a la regió del Souss-Massa del Marroc ha posat al descobert l'existència de diversos llinatges evolutius molt divergents molecularment i amb notables diferències morfològiques. L'estudi del verí de diferents llinatges de L. rufescens i de dues espècies de les Illes Canàries ha demostrat que existeixen diferències respecte a la composició principalment entre L. rufescens i les dues espècies canàries, que aquestes últimes expressen una diversitat menor de paràlegs de la família gènica SicTox, i que l'activitat esfingomielinasa D és en tots els llinatges i espècies estudiades tan elevada com en espècies americanes, les quals s'ha comprovat que poden provocar casos de loxoscelime. / The spider genus Loxosceles Henieken and Lowe, 1832 is mainly distributed across the Americas and Africa. Most of the species show a narrow, circumscribed, distribution, although a few exceptions exist, for example L. rufescens (Dufour 1820), described from Europe. The aim of this thesis is to study the diversity of the genus Loxosceles in the Mediterranean Basin, North Africa and the Canary Islands, and to situate this diversity within a phylogenetic framework. The extensive sampling across the studied areas revealed the existence of hidden diversity. Using mitochondrial data, 11 different evolutionary lineages were identified within L. rufescens, presenting two contrasting phylogeographic patterns: (1) lineages with well-structured populations in Morocco and Iberia, and (2) lineages lacking geographic structure across the Mediterranean Basin. Dating analyses placed main diversification events in the Pleistocene, and multiple Pleistocene refugia, identified using ecological niche modelling (ENM), are compatible with allopatric differentiation of the lineages. Human-mediated transportation appears to have complicated the current biogeography of this medically important and synanthropic spider. This same species was found in the Canary Islands, where phylogenetic analyses revealed the existence of a well-supported clade formed exclusively by Canarian Loxosceles specimens, comprising seven allopatrically distributed evolutionary lineages. Morphological and molecular data confirmed the distinctiveness of six lineages and were described accordingly. Seven newly developed microsatellite markers were used to study the phylogeographic patterns of the species endemic to Fuerteventura and Lanzarote, and the study revealed the importance of sea-level changes and volcanism in shaping the genetic diversity in this species. Also, a new species of Loxosceles was described from Tunisia based on molecular and morphological data. Although this species was first described from a single locality, further sampling across North Africa expanded its distribution from Morocco to Israel. In the Souss-Massa region of Morocco, several highly divergent lineages were detected using molecular data and were shown to be also different morphologically. Using multiple approaches to study venom variation in selected species and lineages from the Mediterranean Basin and the Canary Islands, we found that SMase D activity, the key bioactive component of Loxosceles venom, is comparable to that of the American species that are confirmed to have medically relevant bites.